Dirt Is a Reality

For those who perceive the image of clean, white broilers strutting
about spacious immaculate production houses, we have some unsettling
insights.

Challenge into opportunity Only under highly controlled laboratory setting can birds be this clean.  Dirt is a reality, even if it is not overtly obvious.

For those who perceive the image of clean, white broilers strutting about spacious immaculate production houses, we have some unsettling insights. Only under highly controlled laboratory settings can such a pretty picture be created. Such pristine conditions are quite expensive to maintain and are only practicable for relatively limited numbers of broilers. Perhaps in countries such as Norway, Sweden and New Zealand, where relatively small numbers of birds held under comparatively highly hygienic conditions, are such conditions plausible. In the large-scale broiler producing countries such as the United States, Brazil, China and Thailand (only for example), dirt is a reality.

Even if dirt is not overtly obvious, the lack of biosecurity is obvious to the trained microbiologist. No right-minded person would eat food from the floor.

In contrast, chickens are, by nature, coprophagous and happily dirty. The simplest translation for this characterization is that birds randomly excrete feces in their available living environment, settle onto their keel (breast bone) to rest or sleep (dirtying their feathers and skin) and, happily ingest other birds or their own excreta! Thus, no modern person intentionally employs broilers as pets in one’s home.

Intensively reared birds are grown among flocks numbering 20,000 to 30,000 individuals within commercial production facilities. Hatcheries may produce birds with bacterial pathogens already on or in the chicks. These birds are brought from a hatchery to the broiler house in multiple groups of 100 chicks per plastic flat, which are reared for five to eight weeks within a shelter consisting of a roof and four square walls. This setup provides comparative safety and comfort for the flocks, controlling hawk predation and temperature extremes from the outside environment. Often, dirt floors are covered with litter materials that provide comfort, insulation and absorption for the fecal droppings. Other more modern facilities have poured concrete floors, covered with litter materials. Production houses have a variety of chicken feeds and drinking water readily available to optimize flock weight gain and slaughter weights.

Thus, the environment contains essential elements for the chickens (shelter, food and water) that are also attractive to non-production animals. These uninvited guests might include rodents, insects (such as the Darkling beetle) and wild birds. Bacteria, including pathogens, are co-mingled with these uninvited intruders and serve to seed the flocks, leading to large-scale increases in bacterial numbers. It is the reality of almost inevitable bacterial exposure that is the focus of this manuscript.

Human Pathogens and Poultry
Campylobacter jejuni (and to a lesser extent, E. coli and Salmonella spp. are the main poultry borne agents of human disease associated with poultry. Campylobacter appears to be commensal within the chicken host, but certain isolates of Salmonella can also cause serious animal disease, as seen with S. enteritidis. Understanding the sources and the physiology of these bacteria may enable those involved in poultry production and processing to take measures which reduce the likelihood of disease transmission. 

For example, while hatchery-borne transmission of Salmonella is important, with Campylobacter, the preponderance of available data indicates that vertical transmission rarely, if ever, happens. The opposite to this assertion is that Salmonella can often be cultured from chicks younger than one week post-hatch while, Campylobacter is rarely found in flocks under the age of three weeks. Flocks are often colonized by multiple strains of Campylobacter, while it is infrequent for more than one serotype of Salmonella to be found in a single flock. This would indicate that unrelated repeated breeches of on-farm biosecurity measures occur regularly with Campylobacter transmission.

Both Campylobacter and Salmonella are found widely distributed among a wide array of hosts and, consequently, in water. The transmission of Salmonella in a broiler complex is frequently associated with a single serotype and, this might implicate hatchery-borne transmission as the point source for the broiler chickens. During the cooping and transport of mature flocks to the processing plant Campylobacter numbers on the chicken carcass increase by a thousand fold as compared with on-farm level, while this does not occur with Salmonella because relatively low numbers are colonized in mature birds and the carcasses.

Campylobacter is a relatively fragile bacterium when compared to Salmonella. This is especially important during broiler processing and storage. Campylobacter is very sensitive to even low concentrations of chlorine in chill water; to drying conditions caused by forced air chilling; only moderately warm temperatures kill the organism and freezing conditions consistently reduce numbers by at least tenfold. By comparison, Salmonella is a far more durable organism and can survive such processing interventions.

Campylobacter does not grow outside of the chicken gut and, actually dies during cold storage of chicken carcasses. Salmonella is far more resilient and a single cell is capable of multiplying itself to high numbers under temperature abuse situations. These are important factors as a carcass contaminated with a single cell of Salmonella may multiply and increase the risk of transmission. Higher levels of Campylobacter on carcasses are critically associated with increased probability of disease. Therefore, for this organism, quantification of the level of Campylobacter associated with processed carcasses is far more meaningful than is the mere detection of the organism. 

Salmonella, campylobacter in Humans and Broilers
Internationally, health agencies repeatedly have implicated both Salmonella and Campylobacter in human disease and poultry products are considered as major human health concerns. Poultry-borne transmission of these pathogens has been cited in numerous publications. Sporadic cases of gastroenteritis are most often observed with Campylobacter. However, recent evidence has been presented to suggest that limitations in tracking abilities is the primary explanation that outbreaks are relatively rarely reported. Clusters of human disease associated with poultry-borne transmission was noted in a carefully controlled study conducted in the island of Iceland. Such outbreaks are far more difficult to document among larger populations exposed to far more complex paths of poultry distribution. 

The same strains (either homologous serotypes or homogenous genotypes) are found in both human and broiler flocks. The Centers for Disease Control and Prevention suggests that perhaps 50 per cent of human disease derives from broiler sources. Certainly, because of the itshigh sensitivity of these strains to even low temperatures, mishandling or cross-contamination of poultry products contribute a great deal to this association. In part this is because of the very low infectious dose of Campylobacter, estimated to be as few as 500 cells capable of causing disease. With Salmonella both
sporadic cases and large outbreaks have been widely reported and are associated with poultry products.

Campylobacter is widely distributed in broiler flocks. In the United States, about 85 per cent of all mature flocks are colonized with the pathogen by the time the birds are ready for processing. The levels of intestinal Campylobacter in the processing plant can be greater than 108/gm among more than 90 per cent of the individuals within those colonized flocks. The incidence and the level are primarily dependent upon when the seminal infectious event occurs. In the United States, in the summertime, multiple strains can routinely be cultivated from positive flocks, indicating that the flocks have experienced multiple point sources of infection during that flock cycle. This is quite different from what is observed with Salmonella infection in poultry flocks. Typically, only low levels of Salmonella intestinal colonization are observed at slaughter and the incidence of colonization is also relatively low. Because of its durability, Salmonella, which may have been associated with the birds’ exterior early in life, persist into the processing plant and can be found on the processed carcass, even if the intestinal tract may not be colonized at time of slaughter. Consequently, comparatively low levels of Salmonella are found contaminating processed carcasses.

During broiler carcass processing, the highest numbers of pathogens can be found in post-evisceration, pre-chill operations. Disinfectants are widely applied during water chilling of carcasses. Chill waters can be pH adjusted and also amended with chlorine, TSP, ozone or other antimicrobial disinfectants. These are relatively inexpensive and effective interventions designed to reduce pathogens and spoilage bacteria which otherwise contaminate the processed carcasses. There is considerable and growing consumer disapproval for disinfection of carcasses and, a variety of important countries do not allow such antimicrobials to be added to the chiller waters. However, in the United States the USDA-Food Safety Inspection Service has observed an increasing incidence of Salmonella in processed carcasses over the past several years, despite considerable efforts relating to HACCP interventions. Most often, these HACCP interventions do include the use of processing disinfectants. Increasingly, consumers advocate natural, unaltered products, whichare free of bacterial risks and are free of residues. This is a major challenge for the poultry industry. 

On-Farm Pathogen Control
Economically devastating poultry diseases, such as Newcastle’s, coccidiosis, air-sacculitis and avian influenza occur despite considerable efforts at maintaining biosecurity barriers designed to control these agents. The results of these diseases can be accounted by losses in tens of millions of dollars or in human deaths. Darkling beetles and other vermin exact significant portions of the grower’s profits. Yet, the international poultry industry has not provided surgical suite environments for producing pathogen-free birds while still earning profits. Campylobacter and Salmonella are far less important on the grander scale of things. The industry cannot afford to harvest birds that are uniformly less than 32 days old for the purpose of reducing Campylobacter colonization risks.

As indicated earlier, birds are dirty animals and sources of pathogens as environmental contaminants surrounding production facilities are numerous and cannot practicably be kept out of the production facility. Competitive exclusion – the administering of natural bacterial flora to newly hatched chicks – has been shown to be reasonably successful in the control of Salmonella but has no consistent influence on Campylobacter colonization. Vaccination and phage therapy are potential interventions that are limited by their unacceptably narrow specificities and the constant evolution of bacteria to evade these biological strategies.

Bacteriocins

Campylobacter is widely distributed in broiler flocks. In the United States, about 85 per cent of all mature flocks are colonized with the pathogen by the time the birds are ready for processing.

Bacteriocins are small proteins (polypeptides) produced and secreted by select bacteria. These naturally produced proteins serve to kill heterologous bacteria that are in the ecological proximity of the producing organism by creating a pore (hole) in the target bacteria. Bacteriocin production and activities are a very old bacterial strategy evolved billions of years ago and serve to alter the microflora that may otherwise compete with the producing organism. The producer bacterium has evolved an immunity to the bacteriocin and, therefore, it is impervious to the otherwise lethal function of the polypeptide. The distribution of these antimicrobial polypeptides among bacteria is yet unknown because detection of the bacteriocins still depends upon the target organism and assay used for its detection. If a micro-organism does produce a bacteriocin and the wrong target is used for its detection, its presence will go undetected. As bacteria live within niches containing almost inestimable numbers of competitors, gaining an ecological advantage would likely be necessary for the organism to survive. Advantages might be gained by establishing a strong foothold (such as adherence or invasive capacities), having superior reproductive rates (shorter doubling times), using enhanced or diverse substrate metabolism, or by producing antimicrobial substances. We have evidence to believe that it is through this last approach that enteric bacteria are able to maintain ecological advantage within the intestinal tract. This is where understanding and using bacteriocins can provide agricultural interests with a natural approach to control enteric pathogens. Bacteriocin production and consequent killing of target organisms is an ongoing process within the GI tract of all multi-organ animals (and perhaps in plants as well). Therefore, having a controlled application of bacteriocins might assist the poultry industry in the goal of food safety.

Initially, we were not expressly seeking bacteriocins among a wide variety of intestinal (enteric) bacteria. We were seeking strains of bacteria that could kill or inhibit Campylobacter under in-vitro conditions. Using poultry intestinal materials as our original source, we isolated more than 35,000 bacteria consisting of a variety of genera and species. These isolates were purified and proliferated on agar and, after overnight growth, representative plugs of the growth were placed onto an agar plate containing a lawn of newly spread Campylobacter jejuni, on Campylobacter medium. The plates were incubated for 24 to 48 hours under microaerobic conditions and subsequently zones of inhibition were measured, when present. Among less than 0.5 per cent of the enteric isolates, we observed zones of inhibition of varying sizes. The most promising of the Campylobacter antagonists (creating the largest zones of inhibition) included Gram + spore-forming and lactic acid bacteria (LAB).

These isolates were then grown in broth, the cells were centrifuged from the culture, and the culture was subjected to ammonium sulfate precipitation and subsequent dialysis.

Increased activity units were detected per ml in the crude antimicrobial preparation (CAP). Activity units were determined by twofold dilutions spotted onto Campylobacter medium containing lawns of C. jejuni. At this point we suspected that we were dealing with antimicrobial peptides because of the plating media and extraction procedure we used. Our Campylobacter plating media contains Brucella Agar, which provides only 0.1 per cent of dextrose. Such a low concentration of sugar would not allow production of acids needed to create the zones of inhibition that we saw. In addition, by removing the antagonist cells and by precipitating with ammonium sulfate, we were likely to have a protein-enriched solution after dialysis. 

Next, we subjected the CAP materials to a battery of enzymes and temperature evaluations. Each of the CAP materials were subjected to beta-chymotrypsin, proteinase K and papain protease enzymes, which resulted in destroying the CAP anti-Campylobacter activities. When the CAP materials were subjected to either lysozyme or lipase the activities remained. Likewise, subjecting the CAP materials to temperatures of 90C or greater for 15 minutes left the antimicrobial activities intact. Activities were maintained through pH ranges of 3.0 to 9.1 at high temperatures over 20 minutes of exposure.
Finally, we subjected the CAP to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate polypeptides contained in the solution. The gels were renatured and were overlayed by a semi-soft agar seeded with C. jejuni. The gels and the plates were incubated under microaerobic conditions at 42  degrees (optimal for C. jejuni growth) for about 48 hours and were observed for zones of clearing which surrounded the various bacteriocin polypeptides. A similar assessment was used to determine the isoelectric points of the polypeptides and to demonstrate zones of clearing surrounding the bacteriocins. The estimated molecular weights and the isoelectric points were used to enhance the biochemical purification of the bacteriocins. Finally, the purified bacteriocins were hydrolyzed and the molecular mass was determined by mass spectrometry. The matrix-assisted laser desorption ionization time of flight (MALDI-TOF) system was used. The unique primary amino acid sequence of each bacteriocin was thereby determined.
 
Application of Bacteriocins in Broilers
We have conducted a number of live chicken trials to determine whether we might administer bacteriocins to control concentrations of both Salmonella and Campylobacter in the broiler’s intestine. Using four completely characterized bacteriocins produced by an isolate of Paenibacillus polymyxa, two Enterococcus faecium isolates and, one Lactobacillus salivarius isolate we were repeatedly able to demonstrate reductions of more than one hundred thousand-fold of the target bacteria. Chicken feed emended with 125 mg of E-760 /kg reduced colonization of market age broiler chickens naturally contaminated with Campylobacter spp. Among the untreated 39-day old-broilers the colonization was log10 6.17 cfu Campylobacter spp. per gm of cecal material and, birds given the bacteriocin yielded no detectable (less than 100 cfu per gm) Campylobacter spp. per gram. Likewise, bacteriocin E 50-52 provided in drinking water at levels of 12.5 mg/L reduced Campylobacter spp. from log10 8.00 cfu per gm in the control birds to less than 3.00 cfu per gm of cecal materials in the treated birds. In the same experiment, Salmonella enteritidis was reduced from log10 7.48 cfu per gm of cecal materials in the control bird s vs. non detectable levels in the treated animals. There was also a ten-thousand fold reduction of Salmonella enteritidis in the liver of treated birds when compared to the control bird liver colonization. The minimum inhibitory concentrations of bacteriocin E 50-52 against 98 C. jejuni isolates ranged from 0.025 to 6.4 mg/ml, with the large majority of isolates sensitive to levels below 1.0 mg/ml.  We have published similar, dramatic reductions using both bacteriocins from the P. polymyxa isolate and the L. salivarius isolate. 

Bacteriocins have been FDA approved as human food additives. We have developed fermentations and extraction procedures to produce bacteriocins inexpensively. People have been consuming bacteriocins for millennia in fermented meats and milk without any signs of toxicity. If broilers were given bacteriocins during the last three days of production, both Salmonella and Campylobacter levels would be dramatically reduced and consequently, consumer exposure through processed poultry would correspondingly be reduced. Vaccination of broilers against these pathogens does not seem practicable as there is a wide range of antigenicity among the bacterial groups. Similarly, there is a wide range of susceptibility of lytic phage types among these pathogenic groups of bacteria and the organisms would mutate to evade such treatment. Antibiotic resistance is rampant and becoming ineffective. Regulatory agencies are pulling back authorized use of such antibiotic therapeutics in food animals. Biosecurity as an intervention seems rather pointless in the dirty world of broiler production. Bacteriocins provide dramatic control measures against both Salmonella and Campylobacter in the broiler and, address the consumer desire for natural and safe foods.

Funds for the conduct of studies contained in this manuscript were provided by ISTC project #1720.

U. S. Department of Agriculture, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Athens, Georgia.
State Research Centre for Applied Microbiology and Biotechnology (SRCAMB) Obolensk, Russian Federation.
Presented at the XXIII World Poultry Science Association meeting in Brisbane, Australia